B7shRNA质粒载体构建及其在小鼠心脏移植抗排斥作用研究
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摘要
本课题采用体外构建的B7shRNA质粒载体修饰骨髓来源树突状细胞,通过抑制其CD80、CD86表达以阻断B7/CD28协同刺激通路,从中探讨诱导T淋巴细胞无能的机理;研究经RNAi敲减CD80、CD86后的供体来源树突状细胞在异体心脏移植中对移植物的保护作用及其抗排斥机制。
第一部分 B7shRNA质粒载体构建及其阻断共刺激通路作用体外研究
目的:构建针对小鼠树突状细胞(dendritic cell,DC)表面B7分子的短发夹状RNA(small hairpin RNA,shRNA)质粒载体,探讨其对阻断B7/CD28共刺激通路及诱导异系T淋巴细胞无能的作用。方法:体外培养小鼠骨髓源DC;分别构建针对B7-1(CD80)、B7-2(CD86)的shRNA质粒载体pCD80shRNA、pCD861shRNA、pCD862shRNA,分别转染DC,以流式细胞术初步检测对DC表面抗原CD80、CD86表达的影响;以pCD80shRNA和pCD862shRNA共同构建同时针对CD80、CD86的pB7shRNA质粒载体,转染DC,荧光实时定量PCR及流式细胞术检测转染前后CD80、CD86mRNA及DC表面抗原CD80、CD86、CD11c、MHCⅡ表达,混合淋巴细胞培养观察pB7shRNA转染DC对刺激异系T淋巴细胞增殖能力的影响,荧光实时定量PCR测定混合淋巴细胞培养体系中IL-2mRNA表达水平。结果:pCD80shRNA转染DC,使CD80抗原表达由93.42%下降至31.05%,pCD861shRNA、pCD862shRNA,分别转染DC,使CD86抗原表达由78.70%下降至44.26%、37.90%;以pCD80shRNA和pCD862shRNA成功构建针对CD80、CD86的pB7shRNA质粒载体;B7shRNA干扰DC对CD80、
In this research project, B7 shRNA plasmid vector was constructed and was used to modify myeloid DC in that way which the CD80 and CD86 expressions on it were inhibited to block B7/CD28 co-stimulatory pathway in order to explore the mechanism of T lymphocyte anergy. The protective effect to allograft and anti-rejection mechanism in heart allo-transplantation due to donor derived DCs underwent CD80 and CD86 knock down by RNAi were investigated.Part I Construction of B7shRNA Plasmid Vector and InvitroResearch on its Effect of Blocking B7/CD28 Co-stimulatory PathwayObjective: To construct small hairpin RNA(shRNA) plasmid vector targeting B7 molecular of mice dendritic cells(DC) and analysis its effect on blocking B7/CD28 co-stimulatory pathway and inducing T lymphocytes anergy .Methods: Mice myeloid DC were cultured invitro. shRNA plasmid vector pCD80shRNA、 pCD861shRNA 、 pCD862shRNA were constructed and then transfected DC respectively, correspondly, The change of surface antigen CD80、 CD86 expression levels were measured by flowcytometry. pB7shRNA plasmid vector targeting both CD80 and CD86 was constructed using pCD80shRNA and pCD862shRNA. The mRNA expression level of CD80、 CD86 and the expression level of surface antigen CD80、 CD86、 CD11c、 MHCⅡ were measured by real time quantitative PCR and flowcytometry before and after pB7shRNA transfecting DC. The influence on DC stimulating allogenic T cell proliferation induced by pB7shRNA transfected DC was observed through MLC. IL-2 mRNA levels in MLC were determined by real time
quantitative PCR. Results: The CD80 antigen expressing level declined from 93.42% to 31.05% after the pCD80shRNA transfecting DC, while CD86 declined from 78.70% to 44.26% and 37.90% after the pCD861shRNA and pCD862shRNA transfecting DC respectively. The pB7shRNA plasmid vector targeting both CD80 and CD86 was constructed successfully using pCD80shRNA and pCD862shRNA. After the pB7shRNA transfecting DC, the CD80> CD86 mRNA expression were inhibited at the rate of 77.5% and 61.5% respectively, companying with reducing of the surface antigen CD80+ and CD86+ from 93.42%, 78.70% to 30.05% and 34.76% respectively, but no changing in surface antigen of MHCII, CD lie. The MLC result showed that the index of stimulation to allogenic T cell was declined significantly after the pB7shRNA transfecting DC (P<0.0\), and the IL-2 mRNA levels in MLC was inhibited at the rate of 80.48%. Conclusion: The construction of pB7shRNA plasmid vector applied a stable vector for long-time RNAi research on B7 molecules of mice DC. With the method of pB7shRNA plasmid vector transfecting DC, B7 expression will be silenced efficiently and the B7/CD28 co-stimulatory pathway will be blocked , the ability of DC that stimulate allogenic T lymphocytes proliferation and IL-2 secreation will be knocked down ,thus the T lymphocytes will be induced anergy.Part II The Research on the Anti-rejection Effect Induced byB7shRNA Interfered DC in Mice Heart TVansplantationObjective: To observe the anti-rejection effect of B7shRNA interfered DC in mice heart transplantation and analysis the anti-rejection mechanism. Methods: At first, the heterotopic heart transplantation model in mice was established, then the B7shRNA interfered DC of donors were were transfused into recipients 7 days before operation(group 1: DC treated by shRNA), and the four control groups were:
group2: isograft transplantation, group3: allograft transplantation, group4: treated with CsA, group5: DC none treated with shRNA. The graft survivals were individually recorded and pathological grading of graft rejection were evalued on the 7 day of post-operation. At the same time the IL-2mRNA expression level in grafts were determined by real time quantitative PCR. Results: Stable heterotopic heart transplantation model in mice was established successfully. Comparing with the group3 and group5, the graft survivals were significantly longer in the group of DC treated by shRNA (group 1),(MST: 22 vs 9 and 8 days, P<0.0\ ) companying with lower pathological grade of graft rejection( x2=36.566, PO.01 ) and lower IL-2 mRNA expression level in grafts ( 0.117 + 0. 039 vs 0.376 ±0. 068 and 0.436 ± 0. 055, P<0.0\ ) . There exists positive correlation between the IL-2 mRNA expression level and pathological grade of graft rejection (r=0.931, P <0.01) . Conclusions: Mouse heterotopic heart transplantation model is ideal and valuable for transplantation rejection research. For mice heart transplantation, transfusing donor's B7shRNA interfered DC into recipients before operation can creat anti-rejection effect and the mechanism might be: the blockade of B7/CD28 co-stimulatory pathway by B7shRNA interfered DC will induce the T lymphocytes of recipients anergy. IL-2 might be molecular makers to recognize post-transplantation reaction.
第一部分 B7shRNA质粒载体构建及其阻断共刺激通路作用体外研究
目的:构建针对小鼠树突状细胞(dendritic cell,DC)表面B7分子的短发夹状RNA(small hairpin RNA,shRNA)质粒载体,探讨其对阻断B7/CD28共刺激通路及诱导异系T淋巴细胞无能的作用。方法:体外培养小鼠骨髓源DC;分别构建针对B7-1(CD80)、B7-2(CD86)的shRNA质粒载体pCD80shRNA、pCD861shRNA、pCD862shRNA,分别转染DC,以流式细胞术初步检测对DC表面抗原CD80、CD86表达的影响;以pCD80shRNA和pCD862shRNA共同构建同时针对CD80、CD86的pB7shRNA质粒载体,转染DC,荧光实时定量PCR及流式细胞术检测转染前后CD80、CD86mRNA及DC表面抗原CD80、CD86、CD11c、MHCⅡ表达,混合淋巴细胞培养观察pB7shRNA转染DC对刺激异系T淋巴细胞增殖能力的影响,荧光实时定量PCR测定混合淋巴细胞培养体系中IL-2mRNA表达水平。结果:pCD80shRNA转染DC,使CD80抗原表达由93.42%下降至31.05%,pCD861shRNA、pCD862shRNA,分别转染DC,使CD86抗原表达由78.70%下降至44.26%、37.90%;以pCD80shRNA和pCD862shRNA成功构建针对CD80、CD86的pB7shRNA质粒载体;B7shRNA干扰DC对CD80、
In this research project, B7 shRNA plasmid vector was constructed and was used to modify myeloid DC in that way which the CD80 and CD86 expressions on it were inhibited to block B7/CD28 co-stimulatory pathway in order to explore the mechanism of T lymphocyte anergy. The protective effect to allograft and anti-rejection mechanism in heart allo-transplantation due to donor derived DCs underwent CD80 and CD86 knock down by RNAi were investigated.Part I Construction of B7shRNA Plasmid Vector and InvitroResearch on its Effect of Blocking B7/CD28 Co-stimulatory PathwayObjective: To construct small hairpin RNA(shRNA) plasmid vector targeting B7 molecular of mice dendritic cells(DC) and analysis its effect on blocking B7/CD28 co-stimulatory pathway and inducing T lymphocytes anergy .Methods: Mice myeloid DC were cultured invitro. shRNA plasmid vector pCD80shRNA、 pCD861shRNA 、 pCD862shRNA were constructed and then transfected DC respectively, correspondly, The change of surface antigen CD80、 CD86 expression levels were measured by flowcytometry. pB7shRNA plasmid vector targeting both CD80 and CD86 was constructed using pCD80shRNA and pCD862shRNA. The mRNA expression level of CD80、 CD86 and the expression level of surface antigen CD80、 CD86、 CD11c、 MHCⅡ were measured by real time quantitative PCR and flowcytometry before and after pB7shRNA transfecting DC. The influence on DC stimulating allogenic T cell proliferation induced by pB7shRNA transfected DC was observed through MLC. IL-2 mRNA levels in MLC were determined by real time
quantitative PCR. Results: The CD80 antigen expressing level declined from 93.42% to 31.05% after the pCD80shRNA transfecting DC, while CD86 declined from 78.70% to 44.26% and 37.90% after the pCD861shRNA and pCD862shRNA transfecting DC respectively. The pB7shRNA plasmid vector targeting both CD80 and CD86 was constructed successfully using pCD80shRNA and pCD862shRNA. After the pB7shRNA transfecting DC, the CD80> CD86 mRNA expression were inhibited at the rate of 77.5% and 61.5% respectively, companying with reducing of the surface antigen CD80+ and CD86+ from 93.42%, 78.70% to 30.05% and 34.76% respectively, but no changing in surface antigen of MHCII, CD lie. The MLC result showed that the index of stimulation to allogenic T cell was declined significantly after the pB7shRNA transfecting DC (P<0.0\), and the IL-2 mRNA levels in MLC was inhibited at the rate of 80.48%. Conclusion: The construction of pB7shRNA plasmid vector applied a stable vector for long-time RNAi research on B7 molecules of mice DC. With the method of pB7shRNA plasmid vector transfecting DC, B7 expression will be silenced efficiently and the B7/CD28 co-stimulatory pathway will be blocked , the ability of DC that stimulate allogenic T lymphocytes proliferation and IL-2 secreation will be knocked down ,thus the T lymphocytes will be induced anergy.Part II The Research on the Anti-rejection Effect Induced byB7shRNA Interfered DC in Mice Heart TVansplantationObjective: To observe the anti-rejection effect of B7shRNA interfered DC in mice heart transplantation and analysis the anti-rejection mechanism. Methods: At first, the heterotopic heart transplantation model in mice was established, then the B7shRNA interfered DC of donors were were transfused into recipients 7 days before operation(group 1: DC treated by shRNA), and the four control groups were:
group2: isograft transplantation, group3: allograft transplantation, group4: treated with CsA, group5: DC none treated with shRNA. The graft survivals were individually recorded and pathological grading of graft rejection were evalued on the 7 day of post-operation. At the same time the IL-2mRNA expression level in grafts were determined by real time quantitative PCR. Results: Stable heterotopic heart transplantation model in mice was established successfully. Comparing with the group3 and group5, the graft survivals were significantly longer in the group of DC treated by shRNA (group 1),(MST: 22 vs 9 and 8 days, P<0.0\ ) companying with lower pathological grade of graft rejection( x2=36.566, PO.01 ) and lower IL-2 mRNA expression level in grafts ( 0.117 + 0. 039 vs 0.376 ±0. 068 and 0.436 ± 0. 055, P<0.0\ ) . There exists positive correlation between the IL-2 mRNA expression level and pathological grade of graft rejection (r=0.931, P <0.01) . Conclusions: Mouse heterotopic heart transplantation model is ideal and valuable for transplantation rejection research. For mice heart transplantation, transfusing donor's B7shRNA interfered DC into recipients before operation can creat anti-rejection effect and the mechanism might be: the blockade of B7/CD28 co-stimulatory pathway by B7shRNA interfered DC will induce the T lymphocytes of recipients anergy. IL-2 might be molecular makers to recognize post-transplantation reaction.
引文
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